Calcium measurements in enzymatically dissociated or mechanically microdissected mouse primary skeletal muscle fibers

Summary Here, we provide a protocol for isolation of mouse primary skeletal muscle fibers using two alternative approaches—enzymatic dissociation or mechanical microdissection. We describe the procedures for surgical removal of muscle of interest and isolation of intact single-muscle fibers by either collagenase digestion or mechanical microdissection. We then detail intracellular calcium measurements by microinjecting or loading the isolated muscle fibers with membrane permeable calcium dyes. Finally, we outline steps for intracellular calcium quantification by fluorescent measurement. For complete details on the use and execution of this protocol, please refer to Gineste et al.1


SUMMARY
Here, we provide a protocol for isolation of mouse primary skeletal muscle fibers using two alternative approaches-enzymatic dissociation or mechanical microdissection. We describe the procedures for surgical removal of muscle of interest and isolation of intact single-muscle fibers by either collagenase digestion or mechanical microdissection. We then detail intracellular calcium measurements by microinjecting or loading the isolated muscle fibers with membrane permeable calcium dyes. Finally, we outline steps for intracellular calcium quantification by fluorescent measurement. For complete details on the use and execution of this protocol, please refer to Gineste et al. 1

BEFORE YOU BEGIN
Methods used for cell isolation may result in alterations of the extracellular matrix (ECM), which can impact cellular functionality and affect experimental outcomes in a wide variety of tissues, including liver, 2 lung, 3 bone 4 and skeletal muscle. 5,6 In this protocol, we describe two simplified methods for the isolation of single primary skeletal muscle fibers: 1) enzymatic isolation; and 2) surgical microdissection. We then detail how to use those isolated fibers for calcium measurements. The microdissection method results in improved maintenance of mitochondrial functionality, calcium handling and cellular transcriptomes. The protocol constitutes an adaptation and amalgamation of several enzymatic and microdissection techniques described earlier. [7][8][9] Institutional permissions All animal experiments have to comply with local ethical guidelines and protocols. The protocols used here were approved by the Stockholm North Local Animal Ethics Committee and complied with the Swedish Welfare Ordinance, and applicable regulations and recommendations from Swedish authorities.

MATERIALS AND EQUIPMENT
We herein describe the isolation of FDB muscles of 12 week old female C57BL/6JRj mice.
Note: The protocol is also compatible with the isolation of FDB muscles and from other strains, ages or sexes. Furthermore, it can serve as the starting point for isolation of other murine skeletal muscles beyond the FDB. In such case, depending on the muscle of interest, slight protocol adaptations might be necessary.
Note: Providers might not give an accurate measure of collagenase activity but activities around 280-345 U/mg should give optimal results.
Prepare a 10x solution of Indo-1 AM and pluronic F-127 by dissolving them in DMSO as follows: REAGENT

Reagent Final concentration Amount
Indo-1 AM 0.5 mM 100 mg The solution is to be freshly prepared on the day of the experiment and kept in the dark at room temperature (20 C-25 C).

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STAR Protocols 4, 102260, June 16, 2023 Note: Complete DMEM/F12 and Tyrode solution can be stored at 4 C for up to 1 week.

STEP-BY-STEP METHOD DETAILS
Part I. Surgical removal of FDB muscles

Timing: 30 min
This section describes the surgical removal of the FDB muscle, which serves as the substrate for the single fiber isolations described in the following part II.
1. Sacrifice the animal according to the locally approved procedures. 2. Remove the foot from the leg by amputating at the level of the ankle joint using sharp surgical scissors. 3. Place the foot in prewarmed Tyrode solution. 4. Pin the outermost toes of the isolated foot with dissection needles on the dissection pad with the plantar side of the foot facing upward ( Figure 1D).

Note:
We use the lid of a pipette tip box coated with a 1 cm thick layer of Sylgard 182 elastomer as the dissection pad.
5. Remove the skin overlaying the FDB using dissection scissors. 6. Grip and lift the proximal tendon of the FDB using forceps. 7. Make a transverse incision to separate the FDB tendon from its origin at the back of the heel (calcaneus). 8. Carefully cut and remove all soft and connective tissue between the FDB muscle and the underlying flexor digitorum longus (FDL) using forceps and dissection scissors ( Figure 2A).
CRITICAL: Always point the dissection scissors towards the FDL to minimize the risk of damaging fibers in the FDB.
9. Cut the FDB tendons as far out on the toes as possible to leave a long distal tendon. This helps a lot in the later stages of single fiber dissections.
CRITICAL: Make sure not to stretch the FDB muscle. This is best done by folding back the freed portion of the FDB onto the part of the FDB that remains joined by connective tissue to the underlying FDL muscle. Do not hold or pull the FDB upwards in the solution.
CRITICAL: During the whole procedure, ensure that the muscle does not dry out by covering the foot in warmed Tyrode solution.
Part II. Isolation of single skeletal muscle fibers Note that the following parts IIa and IIb are alternative methods for the isolation of single skeletal muscle fibers. For enzymatic dissociation, follow steps 10-18. For mechanical microdissection, follow steps 19-27.

Timing: 3-4 h
This part describes the isolation of primary single muscle fibers from FDB muscles by enzymatic dissociation.
10. Place 50 mL of complete DMEM/F12 in an incubator at 37 C.

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11. Prepare 5 mL of supplemented collagenase type 1. 12. Clean FDB muscles of tendons, connective tissue, and blood vessels. 13. Incubate the cleaned muscle for 2-3 h at 37 C in the supplemented type 1 collagenase. 14. Transfer muscles to 3 mL fresh DMEM/F12 that was prewarmed in step 10. 15. Gently triturate the muscle using a regular blue plastic 1 mL pipette tip filled with 900 mL DMEM/F12.
Note: Pipette slowly 10-20 times up and down to separate the individual muscle fibers.
Note: One second up and one second down is the right timing.
16. Transfer a volume of 300 mL of the resultant muscle fiber suspension in laminin-coated 35 mm glass-bottom Petri dishes. This part describes the isolation of primary single muscle fibers from FDB muscles by microdissection.
19. Place the FDB muscle in a custom-made dissection tray equipped with a pair of hollow movable metal 10G syringe needles mounted at opposite ends (see setup in Figure 1B). 20. Fix both tendons of the muscle by inserting the proximal and the distal tendons into the split ends of a nylon rod located inside the metal 10G syringe needles ( Figure 2C).

Note:
The nylon rods are pulled into the needles to hold the tendons tightly and the needles can be rotated as needed when cutting away fibers and connective tissue during the dissection.
21. Make FDB fibers accessible by removing connective tissue, fat, and visible blood vessels using sharpened dissection forceps and scissors. 22. Separate the FDB muscle longitudinally into three FDB digits.
Note: It is recommended to use a stereomicroscope with dark-field illumination with up to 403 magnification for this process.
CRITICAL: It is important to keep some muscle fibers of each FDB digit attached to the tendons at both sides (proximal and distal). These are the fibers that will be used for isolation.
23. Mount one digit in the dissection tray and fix into the split rods on both sides ( Figure 2D). 24. Assess whether fibers are susceptible to electrical stimulation by applying single electrical stimuli using a pen stimulator at supramaximal voltage (%10 V).
Note: A brief twitch contraction of a few milliseconds followed by relaxation shows that the fibers are intact and functional (Methods video S1).
CRITICAL: We use a pen-type stimulation electrode connected to a generic current pulse stimulator with up to 100V stimulation voltage (World Precision Instruments; model no. SYS-A300 or Aurora Scientific; model no. 701C).
25. At 403 magnification, select a few twitching fibers that are positioned on the FDB surface and isolate those by cutting and removing all other fibers in small steps ( Figure 2E).
CRITICAL: Check the remaining fibers repeatedly to identify fibers that give a robust contraction upon electrical stimulation.
CRITICAL: Make sure not to damage the fibers by overstretching the FDB digit.

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26. Decide on one fiber and carefully remove the other fibers using the jeweller's forceps and microiris scissors ( Figure 2F). 27. Check that the isolated single fiber contracts upon electrical stimulation.
Part III. Calcium measurements in microdissected muscle fibers For measurements of calcium in enzymatically dissociated fibers, we refer to the interested reader to the accurate methodological descriptions in ref. 10,11 Note that the following parts IIIa and IIIb are alternatives for calcium measurements. In our experience, the best results are obtained with injection of the dye. For injection with intracellular calcium indicator, follow steps 28-34. For loading the fiber with a membrane permeable dye, follow steps 35-37.

Timing: 45 min
This section describes the measurement of calcium in isolated single fibers by microinjection of an intracellular calcium dye.
28. The proximal and distal tendons of the mechanically dissected single fibers are trimmed longitudinally and fitted into aluminum or platinum T-clips using two pairs of forceps to fold the T-clips.
CRITICAL: T-clips should be clamped to the tendon as close as possible to the muscle fiber as a long tendon can interfere with some downstream applications, such as force measurements ( Figure 2G).
CRITICAL: It is essential to have an intact single fiber free of debris when using membrane permeable fluorescent calcium indicators, such as indo-1 AM or fura-2 dyes, because indicator trapped in remnant dead fibers can affect fluorescent measurements resulting in drastically increased experimental variability. For comparison, Figures 2H and 2I show a clean fiber and a fiber that was not well cleaned from connective tissue.
29. Install a micropipette in a micromanipulator and load it with $0.5 mL of 10 mM indo-1 salt solution.
Note: If other calcium dyes are to be used, the concentration might need to be titrated for optimal results.

CRITICAL: Pressure during injection is driven by inert nitrogen gas.
CRITICAL: It is recommended to use Picospritzer gas pressure pulse settings of $100 psi with a duration of 1-5 ms.
30. Mount the fiber in the recording chamber between a force transducer and an adjustable holder ( Figure 2J).
CRITICAL: During this procedure the stimulation chamber should be mounted onto an inverted microscope. Suitable stimulation chambers can be custom-made or are available commercially (e.g., Aurora Scientific; model no. 1500A). Suitable chambers have one mounting peg attached to a force transducer or fixed end and the other mounting peg attached to a movable screw. Superfuse the fiber with Tyrode solution at the desired temperature. For reference, the in vivo temperature of FDB muscles is 31 C (ref. 12 ).

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STAR Protocols 4, 102260, June 16, 2023 31. Adjust the stimulation current intensity and fiber length to obtain maximum tetanic force. a. To this end, determine the supramaximal stimulation intensity by stimulating with 70 Hz tetani at $1 min intervals and increase the voltage incrementally until the stimulation intensity is 10%-20% above that needed to achieve the maximum force. b. Thereafter, adjust the fiber length by using the movable screw to the length that gives maximum tetanic force. 32. Position the tip of the injection micropipette in close proximity to the fiber.
CRITICAL: The emitted fluorescence is measured with any suitable fluorescence system, which, for indo1 AM, has two photomultiplier tubes (e.g., HORIBA, Wedel, Germany or IonOptix, Amsterdam, The Netherlands).
CRITICAL: Measure and subtract background fluorescence of the fiber prior to injection by recording the emitted fluorescence signal values at 405 and 495 nm.

Inject the fiber with the dye.
CRITICAL: It is important to make sure that the fiber is in focus since imprecision can lead to an inaccurate estimate of the amount of injected dye.
CRITICAL: To control the amount of dye injected in the fiber, measure the increase in fluorescence signal at 405 nm-495 nm without stimulating the fiber. Withdraw the pipette when enough dye ($two-times the background fluorescence) has been injected.
34. Allow an even distribution of the dye through the myoplasm of the fiber by waiting for at least 20 min before starting the experimental recordings.
Part IIIb. Loading the fiber with a membrane permeable dye

Time: 1.5 h
This section describes the measurement of calcium in isolated single fibers by loading the fiber with a membrane permeable indicator.
35. Incubate the fiber in the diluted indo-1 AM dye for at least 1 h at room temperature (20 C-25 C) to allow the dye to diffuse into the fiber.
CRITICAL: It is important to avoid that the fiber is drying out during loading in the stimulation chamber. This is done by placing a water filled cap from an Eppendorf tube in the incubation dish and ensuring that the lid is tightly sealed by wrapping it in Parafilm.
36. Mount the fiber as described above. 37. Superfuse with Tyrode solution for $30 min to remove any remaining dye outside of the fiber before starting the measurement.
Part IV. Quantification of calcium measurements

EXPECTED OUTCOMES
The mechanical microdissection method allows isolation of 3 (or up to 6 if each toe is split longitudinally into two) viable single fibers from one FDB whereas as many as 50-100 viable fibers can be isolated when the enzymatic dissociation method is used. Isolated fibers deteriorate in functionality and phenotype, and we do not typically culture them for >24 h. The advantage of isolating intact skeletal muscle fibers by mechanical microdissection is that one can study both contractile function and calcium homeostasis of the muscle fiber within an intact microenvironment. Moreover, the use of single fibers permits metabolic profiling independently of hypoxia or nutritional diffusion limits as is the case when analyzing larger bundles or whole muscles.

LIMITATIONS
The isolation of single skeletal muscle fibers by enzymatic dissociation does not preserve the native microenvironment, which impairs functional and metabolic outcomes. 1 The limitations of microdissection are the technical sophistication and relatively low throughput.

Problem 1
Low viability of isolated single fibers (part I).
Potential solution Make sure to keep longer tendons. Always use the tips of the micro-iris scissors to cut away material. Use only forceps with undamaged tips that meet and close exactly.

Problem 2
The fibers are not contracting (steps 24-27). Problem 3 Difficulties in injecting indicator into the fiber (step 33).

Potential solution
The micro-electrode might be clogged or blocked. Use a new micro-electrode. Increase the Picospritzer gas pressure and/or the duration of the injection pulses.

Potential solution
Increase the time for the indicator to load into the muscle fiber. Be careful to increase the loading time by no more than 15 min as you run the risk of overloading, which will give very smooth records but will incorrectly report time and amplitude of the calcium signals.

Problem 5
Difficulty to visualize the calcium indicators after fiber injection (steps 38-40).

Potential solution
Check that the fiber is illuminated with the excitation light. Replace the dye after >1 month of daily use and repeated freeze-thaw cycles.

Materials availability
All materials are commercially available as indicated.

Data and code availability
This study did not generate any unique datasets or codes.